The Molecular Biology and Gene Knockout Facility will be used by all of the subprojects of this program project. The core will provide services that will greatly facilitate the research proposed by each of these investigators. Core B is located adjacent to the Physiology Department at the University of Wisconsin Biotechnology Center (UWBC). It is a well-equipped laboratory with convenient access to the major services of the Biotechnology Center. Core B is available to all of the subprojects and provides access to state-of-the-art laboratory space and equipment, so that investigators are able to perform certain experiments without borrowing or investing in specialized equipment. In addition, the staff of Core B have extensive experience in molecular biology techniques. Dr. Powers has 19 years of experience as a molecular biologist and geneticist, and Mr. Bartley has over 6 years of experience with molecular biology techniques. Core B staff are continuously available to provide training in molecular biology techniques, consultation on experimental design, troubleshooting of on-going experiments, and assistance in manuscript preparation and grant writing. In addition, because this facility has been assisting members of the Physiology Department for over four years, we have developed expertise in the ongoing projects, and thus can provide in depth assistance to the investigators. Core B will offer several service, because they are required by several subprojects and performing them in a central location will allow the efficient use of equipment, materials and labor. Dr. Powers has already established a successful molecular biology core facility for the Department of Physiology in which all of the following methods are in routine use. The services provides will be: 1) Screening cDNA, genomic bacteriophage, cosmid, and BAC libraries. Characterization of the resulting clones by restriction mapping and DNA sequence analysis. 2) Construction of expression vectors for use in bacterial cells or mammalian cells and the use of in vitro site-directed mutagenesis to create specific mutations in these expression constructs. 3) Engineering of transgene plasmids and the detection of the resulting transgenic founder animals by Southern blotting and PCR. Assistance in establishing the transgenic colony. 4) Engineering of knockout vectors, introduction of the knockout vector into mouse embryonic stem cells, antibiotic selection and identification of correctly targeted ES cell clones by Southern blotting. Assistance in establishing the knockout colony. 5) Design and engineering of vectors used to express recombinant proteins in bacteria or insect cells. 6) Consulting and training in tissue culture techniques for insect cells and murine embryonic stem cells.